Ligand having agonistic activity to mutated receptor

ABSTRACT

The present inventors used antibody engineering techniques to prepare functional antibodies that correspond to individual mutations in causative genes of diseases, and discovered that such antibodies enable the treatment of the diseases. Specifically, the inventors succeeded in preparing ligands, particularly minibodies, which have agonistic activity to receptors that have almost completely lost responsiveness to their natural ligands because of gene mutations (for example, a thrombopoietin (TPO) receptor whose reactivity to TPO has been markedly impaired), and which can transduce signals by interacting with these mutant receptors at levels comparable to normal.

TECHNICAL FIELD

The present invention relates to ligands having agonistic activity tomutant receptors, and pharmaceutical compositions comprising the ligandsas active ingredients.

BACKGROUND ART

In recent years, the causative genes of various diseases have beenidentified in quick succession, and a variety of therapeutic methods forsuch diseases have been studied and established. Of these methods, themost intensively studied are mostly therapeutic methods forcomplementing enzyme gene deficiencies. It has been reported that enzymereplacement therapy using “Cerezyme” (Genzyme) is effective for patientswith Gaucher's disease, in which β-glucocerebrosidase is deficient, andthat enzyme replacement therapy using “Aldurazyme” (Genzyme) iseffective for patients with mucopolysaccharidosis, in whichα-L-iduronidase is deficient. Previously attempted gene therapiesinclude introducing the adenosine deaminase (ADA) gene to patients withADA deficiency, and introducing the coagulation factor IX gene topatients with hemophilia B. In addition to enzyme deficiencies, a largenumber of genetic diseases are known, such as genetic diseases ofcytokines and their receptors. Some patients with type II diabetesmellitus, which accounts for approximately 90% of diabetes mellituscases, have been reported to have insulin receptor deletions ormutations. Such deletions and mutations are assumed to cause thedisease. Furthermore, some patients with thrombocytopenia have beenreported to have thrombopoietin receptor deletions and mutations, andthe failure of TPO signaling can be thought to cause the disease. Todate, no fundamental therapeutic methods have been available for suchgenetic diseases, and the establishment of such therapeutic methods isexpected.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorderthat causes thrombocytopenia in infancy and pancytopenia in laterchildhood. It has been revealed that TPO, a thrombopoietic growthfactor, is present in CAMT patients at a high concentration in sera, butthat platelets and hematopoietic precursor cells lack TPOresponsiveness. Most of these patients have been found to carry pointmutations in their thrombopoietin receptor (c-MPL) gene. It has alsobeen reported that such mutations result in frame shift or insertion ofa termination codon, leading to patients who have a total loss ofthrombopoietin receptor function and patients who have a homozygous orheterozygous amino acid substitution in the extracellular domain of thereceptor (see Non-patent Document 1). Bone marrow transplant is the onlycurrently available therapeutic method for treating such patients.

[Non-Patent Document 1]

Matthias Ballmaier, Manuela Germeshausen, Harald Schulze, KlaraCherkaoui, Sabine Lang, Annika Gaudig, Stephanie Krukemeier, MartinEilers, Gabriele Strausz, and Karl Welte, “BLOOD”, vol. 97, No. 1, pp.139 (Jan. 1, 2001).

DISCLOSURE OF THE INVENTION

The present invention was achieved in view of the circumstancesdescribed above. An objective of the present invention is to provideligands having agonistic activity to mutant receptors.

The present inventors studied intensively to achieve the objectivedescribed above. By using antibody engineering techniques, the inventorsprepared functional antibodies corresponding to each of the mutations inthe causative genes of the diseases described above, discovering thatthese antibodies were useful to treat such diseases. Specifically, theinventors succeeded in preparing minibodies, each of which has agonisticactivity to a mutant receptor that has almost completely lostresponsiveness to its natural ligand due to gene mutation (for example,a thrombopoietin (TPO) receptor whose responsiveness to TPO is mostlylost), and which can transduce signals at levels comparable to normallevels when reacted with such a mutant receptor.

Diseases caused by gene mutations can be treated by using suchantibodies or modified products thereof. Alternatively, such diseasescan be treated by gene therapy using genes capable of expressing theantibodies or modified products thereof.

CAMT patients, who were previously difficult to treat, can now betreated using ligands having agonistic activity, such as the antibodiesof the present invention or the genes encoding them.

In addition, the methods of the present invention are applicable tovarious other diseases caused by mutations in genes encoding receptorson cell membranes. Thus, the present invention can provide newtherapeutic methods for these diseases.

Specifically, the present invention relates to ligands having agonisticactivity to mutant receptors, more specifically provides:

[1] a ligand having agonistic activity to a mutant receptor;

[2] the ligand of [1], where the ligand is an antibody;

[3] the ligand of [1] or [2], where the ligand has greater agonisticactivity to the mutant receptor than the natural ligand;

[4]. the ligand of any one of [1] to [3], where the mutant receptor is areceptor resulting from a mutation(s) in the amino acid sequence;

[5] the ligand of any one of [1] to [4], where the mutant receptor haslost responsiveness to the natural ligand;

[6] the ligand of any one of [1] to [5], where the mutant receptorcauses a disease;

[7] the ligand of any one of [1] to [6], where the mutant receptor is amutant thrombopoietin receptor;

[8] the ligand of [2], where the antibody is a minibody;

[9] the ligand of [8], where the minibody is a diabody;

[10] a method for transducing a signal to a mutant receptor by binding aligand;

[11] the method of [10], where the ligand is an antibody;

[12] the method of [10] or [11], where the mutant receptor results froman amino acid mutation(s);

[13] the method of any one of [10] to [12], where the mutant receptorhas lost responsiveness to the natural ligand;

[14] the method of any one of [10] to [13], where the mutant receptor isassociated with disease onset;

[15] the method of any one of [10] to [14], where the mutant receptor isa mutant thrombopoietin receptor;

[16] a method for treating a disease caused by a mutant receptor, bybinding a ligand to the mutant receptor;

[17] the method of [16], where the ligand is an antibody;

[18] a method of screening for a ligand having agonistic activity to amutant receptor, where the method comprises the steps of,

(a) contacting a test substance with the mutant receptor,

(b) detecting a signal in the mutant receptor, and

(c) selecting a ligand having agonistic activity;

[19] a method of screening for a ligand having agonistic activity to amutant receptor, where the method comprises the steps of,

(a) determining agonistic activity to a normal receptor,

(b) determining agonistic activity to the mutant receptor, and

(c) selecting a ligand having greater agonistic activity to the mutantreceptor than the normal receptor;

[20] a method of screening for a ligand having agonistic activity tonormal and mutant receptors, where the method comprises the steps of,

(a) determining agonistic activity to the normal receptor,

(b) determining agonistic activity to the mutant receptor, and

(c) selecting a ligand having agonistic activity to both normal andmutant receptors;

[21] the method of any one of [18] to [20], where the ligand is anantibody;

[22] a substance obtained by the method of any one of [18] to [21];

[23] a therapeutic agent for a disease caused by a mutant receptor,where the agent comprises a ligand of the mutant receptor;

[24] the therapeutic agent of [23], where the ligand is the ligand ofany one of [1] to[9];

[25] the therapeutic agent of [23], where the ligand is an antibody;

[26] the therapeutic agent of any one of [23] to [25], where the mutantreceptor results from an amino acid mutation(s);

[27] the therapeutic agent of any one of [23] to [26], where the mutantreceptor has lost responsiveness to the natural ligand;

[28] the therapeutic agent of any one of [23] to [27], where the mutantreceptor is a mutant thrombopoietin receptor; and,

[29] the therapeutic agent of any one of [23] to [28], where the diseaseis congenital amegakaryocytic thrombocytopenia.

The present invention provides ligands having agonistic activity tomutant receptors.

Mutant receptors of the present invention are usually receptors thatexist at a frequency of less than 50%, preferably less than 20%, morepreferably less than 10%, and even more preferably less than 1%. Thefrequency is generally calculated using randomly selected subjects.However, the frequency may vary depending on country, area, sex, andsuch. Therefore, the frequency may also be calculated, for example,within a selected country or area such as Japan, the United States, orEurope, or be calculated for one sex. When there are two or moremutation sites in a receptor, the frequency may be calculated formultiple mutation sites or for any one of the mutation sites. Mutantreceptors are preferably evaluated using frequency, as described above.However, mutant receptors can also be evaluated, for example, by theirsignal transducing ability. Specifically, for example, when twodifferent receptors are present, the one with stronger transducingsignals upon natural ligand-binding may be used as a non-mutantreceptor, and the other with weaker transducing signals may be used as amutant receptor.

Preferred mutant receptors of the present invention include, but are notlimited to, receptors resulting from amino acid mutations (receptorswith mutated amino acid sequences); however, any type of mutation isacceptable, as long as the mutated receptor influences responsiveness tonatural ligands, or the conformation, sugar chain structure, or spatialrelationship or angles when a receptor exists as a multimer, and so on.Mutations in the amino acid sequence include amino acid substitutions,deletions, insertions, and additions. The receptors of the presentinvention have preferably lost responsiveness to the natural ligands.

Herein, “ligand” refers to a substance that specifically binds to afunctional protein. The type of ligand is not limited. Such ligandsinclude low-molecular-weight compounds, proteins, and peptides. In thepresent invention, functional proteins are preferably receptors. In thepresent invention, ligands preferably have agonistic activity. Thepresent invention also provides methods for transducing signals to amutant receptor by binding a ligand of the present invention. Suchligands for use in the methods of the present invention are preferablynon-natural ligands, and not natural ligands.

In the present invention, it is preferred to target a mutant receptorwhose responsiveness to a natural ligand is different from that of anon-mutant receptor. A “mutant receptor whose responsiveness to anatural ligand is different from that of a non-mutant receptor” refersto a mutant receptor that exhibits agonistic activity and signalingactivity that differs from the activities of the non-mutant receptorwhen the mutant and non-mutant receptors bind to the same natural ligandunder the same conditions. In general, agonistic activity and signalingactivity in mutant receptors is impaired compared with non-mutantreceptors (the mutants have lost their responsiveness to naturalligands).

The receptors include receptors belonging to the hematopoietic growthfactor receptor family, the cytokine receptor family, the tyrosinekinase receptor family, the serine/threonine kinase receptor family, theTNF receptor family, the G protein-coupled receptor family, theGPI-anchored receptor family, the tyrosine phosphatase receptor family,the adhesion factor family, and the hormone receptor family. There aremany documents that describe receptors belonging to these receptorfamilies, and their characteristics; for example Cooke B A, King R J B,van der Molen H J Eds. New Comprehensive Biochemistry Vol.18B “Hormonesand their Actions Part II” pp. 1-46 (1988) Elsevier Science PublishersBV, New York, USA; Patthy L. (1990) Cell, 61: 13-14; Ullrich A. et al.(1990) Cell, 61: 203-212; Massagul J. (1992) Cell, 69: 1067-1070;Miyajima A. et al. (1992) Annu. Rev. Immunol., 10: 295-331; Taga T. andKishimoto T. (1992) FASEB J., 7: 3387-3396; Fantl W I. et al. (1993)Annu. Rev. Biochem., 62: 453-481; Smith C A., et al. (1994) Cell, 76:959-962; Flower D R. (1999) Biochim. Biophys. Acta, 1422: 207-234; CellTechnology: supplementary vol. Handbook series “Handbook for Adhesionfactors” M. Miyasaka Ed. (1994) Shujunnsha, Tokyo, Japan, and so on.Specific receptors belonging to the families listed above include: humanand mouse erythropoietin (EPO) receptors, human and mousegranulocyte-colony stimulating factor (G-CSF) receptors, human and mousethrombopoietin (TPO) receptors, human and mouse insulin receptors, humanand mouse Flt-3 ligand receptors, human and mouse platelet-derivedgrowth factor (PDGF) receptors, human and mouse interferon (IFN)-α and-β receptors, human and mouse leptin receptors, human and mouse growthhormone (GH) receptors, human and mouse interleukin (IL)-10 receptors,human and mouse insulin-like growth factor (IGF)-I receptors, human andmouse leukemia inhibitory factor (LIF) receptors, and human and mouseciliary neurotrophic factor (CNTF) receptors (hEPOR: Simon, S. et al.(1990) Blood 76, 31-35; mEPOR: D'Andrea, AD. Et al. (1989) Cell 57,277-285; hG-CSFR: Fukunaga, R. et al. (1990) Proc. Natl. Acad. Sci. USA.87, 8702-8706; mG-CSFR: Fukunaga, R. et al. (1990) Cell 61, 341-350;hTPOR: Vigon, I. et al. (1992) 89, 5640-5644; mTPOR: Skoda, R C. Et al.(1993) 12, 2645-2653; hInsR: Ullrich, A. et al. (1985) Nature 313,756-761; hFlt-3: Small, D. et al. (1994) Proc. Natl. Acad. Sci. USA. 91,459-463; hPDGFR: Gronwald, R G K. Et al. (1988) Proc. Natl. acad. Sci.USA. 85, 3435-3439; hIFN α/β R: Uze, G. et al. (1990) Cell 60, 225-234,and Novick, D. et al. (1994) Cell 77, 391-400).

In one embodiment, the mutant receptors of the present inventioncomprise receptors associated with disease onset. The phrase “mutantreceptors associated with disease onset” means that the loss ofresponsiveness to a natural ligand is part of the reason that diseaseonset is triggered. In the present invention, a mutant receptor is notnecessarily the sole factor triggering disease onset, and may be acontributing factor. Many reports have been previously publisheddescribing the association of mutant receptors with disease onset;however, in addition to previously reported associations, associationsof mutant receptors and disease onset can also be identified by methodsof statistical analysis (for example, correlation analyses). Correlationanalyses, also called “case control studies”, are well known to thoseskilled in the art (for example, Nishimura, Y., 1991, “Statisticalanalysis of polymorphisms”, Saishin Igaku, 46:909-923; Oka, A. et al.,1990, Hum. Mol. Genetics 8, 2165-2170; Ota, M. et al., 1999, Am. J. Hum.Genet. 64, 1406-1410; Ozawa, A. et al., 1999, Tissue Antigens 53,263-268). For example, the correlation between a mutant receptor and adisease can be studied by determining the frequency of the mutantreceptor in patients and in healthy subjects, and examining whether thepatient population has a higher mutant receptor frequency. Typically,differences in the frequency are evaluated using the x-test. x isobtained by the equation x²=Σ(observed value−expected value)²/expectedvalue. A p value is obtained from the x² value thus determined. Whethera mutant receptor correlates with a disease can be determined based onthis p value. For example, when p<0.05, a mutant receptor is consideredto correlate with a disease.

There are many reports on mutant receptors known to be involved indisease onset. Such mutant receptors specifically include: mutantthrombopoietin (TPO) receptors, mutant insulin receptors, mutanterythropoietin receptors, mutant growth hormone receptors, mutant commonγ chain receptors (common receptor of IL-2, IL-4, IL-7, IL-15, andIL-21), mutant androgen receptors (Glutamine Repeats andNeurodegenerative Disease: Molecular Aspects (2001), 261-267, OxfordUniversity press), mutant receptors for proopiomelanocortin (POMC) andmelanocortin (Journal of Clinical Endocrinology and Metabolism (2001),86(4), 1442-1446), mutant ryanodine receptors (Human Mutation (2000),15(5), 410-417), mutant thyroid-stimulation hormone receptors (Trends inEndocrinology and Metabolism (1998), 9(4), 133-140), and mutantthyrotropin receptors (European Journal of Medical Research (1996),1(10), 460-464). In the present invention, particularly preferred mutantreceptors are mutant thrombopoietin receptors.

Herein, a “natural ligand” refers to a ligand in the body, and ispreferably a ligand with the most influence on non-mutant receptorsignaling. Normally, the natural ligands of the present invention do notcomprise antibodies.

“Agonistic activity” refers to the activity of inducing a change in acertain physiological activity, caused by transducing signals into cellsupon the binding of a ligand to a receptor. The physiological activityincludes, but is not limited to, growth-promoting activity, survivalactivity, differentiation activity, transcription activity, membranetransport activity, binding activity, proteolytic activity,phosphorylation/dephosphorylation activity, oxidation/reductionactivity, transfer activity, nucleolytic activity, and dehydrationactivity.

In the present invention, any detection indicator may be used to assayphysiological activities, as long as it can measure quantitative and/orqualitative change. For example, it is possible to use cell-free assayindicators, cell-based assay indicators, tissue-based assay indicators,and in vivo assay indicators. Indicators that can be used in cell-freeassays include enzymatic reactions and quantitative and/or qualitativechanges in proteins, DNAs, or RNAs. Such enzymatic reactions includeamino acid transfers, sugar transfers, dehydrations, dehydrogenations,and substrate cleavages. Alternatively, the followings can be used:protein phosphorylations, dephosphorylations, dimerizations,multimerizations, catabolisations, dissociations and such; and DNA orRNA amplifications, cleavages, and extensions. For example, proteinphosphorylation downstream of a signal transduction pathway may be usedas a detection indicator. As indicators in cell-based assays, changes tocell phenotype, for example, quantitative and/or qualitative changes inproducts, changes in proliferation activity, morphological changes, orchanges in cellular properties can be used. Products include secretoryproteins, surface antigens, intracellular proteins, and mRNAs.Morphological changes include changes in process formation and/orprocess number, changes in cell flatness, changes in the degree ofelongation/horizontal to vertical ratio, changes in cell size, changesin intracellular structure, heterogeneity/homogeneity of cellpopulations, and changes in cell density. Such morphological alterationscan be observed under a microscope. Cellular properties to be usedinclude anchorage dependency, cytokine-dependent responsiveness, hormonedependency, drug resistance, cell motility, cell migration activity,pulsatility, and alteration in intracellular substances. Cell motilityincludes cell infiltration activity and cell migration activity. Changesin intracellular substances include changes in enzyme activity, mRNAlevels, levels of intracellular signaling molecules such as Ca²⁺ andcAMP, and intracellular protein levels. Furthermore, changes in the cellproliferation activity induced by receptor stimulation can be used as anindicator. The indicators to be used in tissue-based assays includefunctional changes for the tissue in use. Indicators that can be usedfor in vivo assays include changes in tissue weight, changes in theblood system (for example, changes in the number of hemocytes, proteincontents, or enzyme activities), changes in electrolyte levels, andchanges in the circulatory system (for example, changes in bloodpressure or heart rate).

The methods for measuring such detection indicators are not particularlylimited. For example, luminescence, color development, fluorescence,radioactivity, fluorescence polarization values, surface plasmonresonance signals, time-resolved fluorescence, mass, absorptionspectrums, light scattering, and fluorescence resonance energy transfersmay be used. These measurement methods are known to those skilled in theart and may be appropriately selected depending on the purpose. Forexample, absorption spectra can be measured using a conventionalphotometer, plate reader, or such; luminescence can be measured with aluminometer or such; and fluorescence can be measured with a fluorometeror such. Mass can be determined with a mass spectrometer. Radioactivitycan be determined with a measurement device such as a gamma counter,depending on the type of radiation. Fluorescence polarization values canbe measured using BEACON (TaKaRa). Surface plasmon resonance signals canbe obtained using BIACORE. Time-resolved fluorescence, fluorescenceresonance energy transfer, or such can be measured with ARVO or such.Furthermore, a flow cytometer can also be used for measuring. In thepresent invention, it is possible to use a chimeric receptor comprisingan extracellular domain of a mutant receptor and a cytoplasmic domain ofanother protein. For example, when the cytoplasmic domain of G-CSFreceptor, EPO receptor, EGF receptor, or thrombopoietin receptor isused, the cell proliferation activity induced by stimulating thereceptor can be used as a detection indicator. In assays using cellproliferation activity as a detection indicator, cell lines that die inthe absence of ligands are preferably used to improve detectionsensitivity. Cytokine-dependent cell lines are particularly preferredbecause they can be easily passaged. For example, it is possible to useCTLL-2 cells, which are an IL-2-dependent cell line, and 32D cells,FDC-P1 cells, and Ba/F3 cells, which are IL-3-dependent cell lines.These cell lines will die two or three day after the start of culturewhen a cytokine such as IL-2 or IL-3 that is essential for cellproliferation, is eliminated from the culture media. It is preferable touse FDC-P1 cells and Ba/F3 cells that express a chimeric receptorcomprising the cytoplasmic domain of mouse G-CSF receptor.

The ligands of the present invention that have agonistic activity arenot particularly limited, as long as they have agonistic activity tomutant receptors. Such ligands may have agonistic activity to bothmutant and non-mutant receptors, or may only have agonistic activity tomutant receptors. When the ligands have agonistic activity to bothmutant and non-mutant receptors, they may have greater agonisticactivity to the non-mutant receptor, or greater agonistic activity tothe mutant receptor. Alternatively, the ligands may have comparableagonistic activity to the non-mutant and mutant receptors. Nonetheless,when the chief purpose is to treat a disease caused by a mutantreceptor, it is preferable to use a ligand with greater agonisticactivity to the mutant receptor than the non-mutant receptor.

The antibodies of the present invention are not particularly limited, aslong as they have agonistic activity, and mouse antibodies, ratantibodies, rabbit antibodies, sheep antibodies, camel antibodies,chimeric antibodies, humanized antibodies, human antibodies, and suchcan be appropriately used. Such antibodies may be polyclonal ormonoclonal antibodies. Monoclonal antibodies are preferred because theycan be stably produced as homogeneous antibodies. Both polyclonal andmonoclonal antibodies can be prepared by methods known to those skilledin the art.

Hybridomas producing monoclonal antibodies can basically be prepared bythe conventional methods described below. Specifically, immunization iscarried out by a conventional immunization method, using a desiredantigen or cells expressing the desired antigen as a sensitizingantigen. The prepared immunocytes are fused with known parental cells bya conventional cell fusion method. The fused cells are screened formonoclonal antibody-producing cells (hybridomas) by conventionalscreening methods.

The type of sensitizing antigen to be used is not limited. For example,a full-length protein of a receptor of interest, or a partial peptidethereof (for example, an extracellular domain) can be used. The antigenscan be prepared by methods known to those skilled in the art; forexample, according to methods using baculovirus (for example, see WO98/46777). Hybridomas can be prepared, for example, by the method ofMilstein et al. (Kohler, G. and Milstein, C., Methods Enzymol., 1981,73, 3-46.). When an antigen has weak immunogenicity, the antigen may beconjugated with a large immunogenic molecule such as albumin, to achieveimmunization. In addition, the present invention may use recombinantantibodies, produced by gene engineering. The genes encoding theantibodies are cloned from hybridomas, inserted into an appropriatevector, and then introduced into a host (see, e.g., Carl, A. K.Borrebaeck, James, W. Larrick, Therapeutic Monoclonal Antibodies,Published in the United Kingdom by Macmillan Publishers Ltd, 1990).Specifically, using a reverse transcriptase, cDNAs encoding the variableregions (V regions) of the antibodies are synthesized from the mRNAs ofthe hybridomas. DNAs encoding the variable regions of the antibodies ofinterest are obtained, and ligated with DNAs encoding desired constantregions (C regions) of the antibodies, and these constructs are insertedinto expression vectors. Alternatively, DNAs encoding the variableregions of the antibodies may be inserted into expression vectorscomprising the DNAs of the antibody C regions. Those cDNAs are insertedinto expression vectors such that the genes are expressed under theregulation of an expression regulatory region, for example, an enhancerand promoter. Host cells are then transformed using the expressionvectors, and the antibodies can be expressed. The epitopes on themolecules that are recognized by antibodies of the present invention arenot limited to particular epitopes. The antibodies may recognize anyepitope on the molecules. Typically, the antibodies recognize epitopesin the extracellular domain. In the present invention, recombinantantibodies artificially modified to reduce heterologous antigenicityagainst humans can be used. Examples of such recombinant antibodiesinclude chimeric antibodies and humanized antibodies. These modifiedantibodies can be produced using known methods. A chimeric antibodycomprises heavy chain and light chain variable regions of an antibodyfrom a nonhuman mammal such as a mouse, and heavy chain and light chainconstant regions of a human antibody. Such an antibody can be obtainedby (1) ligating a DNA encoding a variable region of a mouse antibody toa DNA encoding a constant region of a human antibody; (2) inserting theresulting construct into an expression vector; and (3) introducing thevector into a host for production of the antibody. A humanized antibody,which is also called a reshaped human antibody, is obtained bytransferring a complementarity determining region (CDR) of an antibodyof a nonhuman mammal such as a mouse, to the CDR of a human antibody.Conventional genetic recombination techniques for the preparation ofsuch antibodies are known. Specifically, a DNA sequence designed toligate a CDR of a mouse antibody with the framework regions (FRs) of ahuman antibody is synthesized by PCR, using several oligonucleotidesconstructed to comprise overlapping portions at their ends. A humanizedantibody can be obtained by (1) ligating the obtained DNA to a DNA thatencodes a human antibody constant region; (2) inserting the resultingconstruct into an expression vector; and (3) introducing the vector intoa host to produce the antibody (see European Patent Application No. EP239,400, and International Patent Application No. WO 96/02576). Humanantibody FRs ligated via the CDR are selected where the CDR forms afavorable antigen-binding site. As necessary, amino acids in theframework region of an antibody variable region may be substituted suchthat the CDR of a reshaped human antibody forms an appropriateantigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).Methods for obtaining human antibodies are also known. For example,desired human antibodies with antigen-binding activity can be obtainedby (1) sensitizing human lymphocytes with antigens of interest or cellsexpressing antigens of interest in vitro; and (2) fusing the sensitizedlymphocytes with human myeloma cells such as U266 (see Japanese PatentApplication Kokoku Publication No. (JP-B) H01-59878 (examined, approvedJapanese patent application published for opposition)). Alternatively,the desired human antibodies can also be obtained by using antigens ofinterest to immunize transgenic (Tg) animals comprising a partial orentire repertoire of human antibody genes (see International PatentApplication WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO96/34096, and WO 96/33735). Furthermore, techniques to obtain humanantibodies by panning with a human antibody library are known. Forexample, the variable regions of human antibodies are expressed assingle chain antibodies (scFvs) on the surface of phages, using a phagedisplay method, and the phages that bind to the antigen can be selected.By analyzing the genes of selected phages, the DNA sequences encodingthe variable regions of human antibodies that bind to the antigen can bedetermined. If the DNA sequences of scFvs that bind to the antigen areidentified, appropriate expression vectors comprising these sequencescan be constructed to obtain human antibodies. Such methods are alreadywell known (see WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO93/19172, WO 95/01438, and WO 95/15388). When the antibody genes areisolated and introduced into appropriate hosts to produce antibodies,hosts and expression vectors can be used in appropriate combinations.Eukaryotic host cells that can be used are animal cells, plant cells,and fungal cells. The animal cells include: (1) mammalian cells such asCHO, COS, myeloma, baby hamster kidney (BHK), HeLa, and Vero cells; (2)amphibian cells such as Xenopus oocytes; or (3) insect cells such assf9, sf2l, and Tn5. Known plant cells include cells derived from theNicotiana genus such as Nicotiana tabacum, which can be callus-cultured.Known fungal cells include yeasts such as the Saccharomyces genus, forexample Saccharomyces cerevisiae, and filamentous fungi such as theAspergillus genus, for example Aspergillus niger. Prokaryotic cells canalso be used in production systems that utilize bacterial cells. Knownbacterial cells include E. coli and Bacillus subtilis. The antibodiescan be obtained by introducing the antibody genes of interest into thesecells by transformation, and then culturing the transformed cells invitro.

The antibodies may be minibodies or modified products of antibodies, aslong as they can bind to antigens. In the present invention, a minibodycomprises an antibody fragment obtained by deleting a portion from awhole antibody (for example, whole IgG). There is no limitation on thetype of minibody, as long as it has the ability to bind to an antigen.The antibody fragments of the present invention are not particularlylimited, as long as they are portions of whole antibodies. The antibodyfragments preferably comprise a heavy chain variable region (VH) or alight chain variable region (VL), and particularly preferably compriseboth a VH and VL. Specifically, the antibody fragments include Fab,Fab′, F(ab′)₂, Fv, and scFv (single-chain Fv). A preferred antibodyfragment is scFv (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A.(1988) 85, 5879-5883; and Plickthun “The Pharmacology of MonoclonalAntibodies” Vol. 113, Eds. Resenburg and Moore, Springer Verlag, NewYork, pp. 269-315, (1994)). Such an antibody fragment can be prepared bytreating an antibody with an enzyme (for example, papain or pepsin) orby inserting a gene construct encoding the antibody fragment into anexpression vector and expressing it in appropriate host cells (see, forexample, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M.and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A.and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E.,Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., MethodsEnzymol. (1986) 121, 663-669; and Bird, R. E. and Walker, B. W., TrendsBiotechnol. (1991) 9, 132-137). The minibodies of the present inventionpreferably have a smaller molecular weight than whole antibodies.However, the minibodies may form multimers (for example, dimers,trimers, or tetramers), and thus their molecular weights can be greaterthan those of whole antibodies.

Preferred minibodies of the present invention comprise two or moreantibody VHs and two or more antibody VLs, in which each of the variableregions are directly linked, or indirectly linked together via linkersor such. The linkages may be covalent or non-covalent bonds, or compriseboth covalent and non-covalent bonds. More preferred minibodies areantibodies comprising two or more VH-VL pairs formed via non-covalentbonding between VH and VL. The distance between the two VH-VL pairs in aminibody is preferably less than that in the whole antibody.

Particularly preferred minibodies of the present invention are diabodiesand sc(Fv)2. A diabody is a dimerized fragment in which two variableregions are linked together via a linker or such (for example, scFv)(hereinafter, referred to as “a fragment constituting a diabody”), andwhich typically comprises two VL and two VH (P. Holliger et al., Proc.Natl. Acad. Sci. USA, 90, 6444-6448 (1993), EP 404097; WO 93/11161;Johnson et al., Methods in Enzymology, 203, 88-98, (1991); Holliger etal., Protein Engineering, 9, 299-305, (1996); Perisic et al., Structure,2, 1217-1226, (1994); John et al., Protein Engineering, 12(7), 597-604,(1999); Holliger et al., Proc. Natl. Acad. Sci. USA. 90, 6444-6448,(1993); Atwell et al., Mol. Immunol. 33, 1301-1312, (1996)). The linksbetween fragments constituting a diabody may be non-covalent or covalentbonds, and are preferably non-covalent bonds.

Alternatively, two fragments constituting a diabody can be linkedtogether via a linker to form a single-chain diabody (scDiabody). Whenthe fragments constituting the diabody are linked together using a longlinker, comprising approximately 20 amino acids, it is possible to linkthe fragments constituting the diabody in the same chain using anon-covalent bond, forming a dimer.

Fragments constituting diabodies include a VL and VH linked together, aVL and VL linked together, a VH and VH linked together, and the like. AVH and VL linked together is preferred. There is no limitation on thetype of linker for linking a variable region and variable region in afragment constituting a diabody. However, it is preferable to use alinker short enough to prevent formation of a non-covalent bond betweenvariable regions in the same fragment. Those skilled in the art canappropriately select the length of such linkers; however, their lengthis typically 2 to 14 amino acids, preferably 3 to 9 amino acids, andparticularly preferably 4 to 6 amino acids. In these cases, the linkerbetween the VL and VH encoded by the same fragment is short, and thus nonon-covalent bonds are formed between VL and VH on the same chain. Thus,a single-chain V region fragment is not formed, and the VL and VH formdimers with other fragments via non-covalent bonds. Further, based onthe same principle for producing diabodies, a multimerized antibody suchas a trimer or tetramer can be prepared by linking three or morefragments constituting a diabody.

The sc(Fv)2 of the present invention are single-chain minibodiesproduced by linking two VHs and two VLs with linkers and such (Hudson etal., 1999, J Immunol. Methods 231:177-189). sc(Fv)2 exhibit aparticularly high agonistic activity compared to whole antibodies andother minibodies. sc(Fv)2 can be produced, for example, by linking scFvmolecules with a linker.

In a preferable antibody, two VHs and two VLs are arranged in the orderof VH, VL, VH, and VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]),beginning from the N terminus of a single-chain polypeptide.

The order of the two VHs and two VLs is not limited to the abovearrangement, and they may be arranged in any order. Examples ofarrangements are listed below:

-   [VL]-linker-[VH]-linker-[VH]-linker-[VL]-   [VH]-linker-[VL]-linker-[VL]-linker-[VH]-   [VH]-linker-[VH]-linker-[VL]-linker-[VL]-   [VL]-linker-[VL]-linker-[VH]-linker-[VH]-   [VL]-linker-[VH]-linker-[VL]-linker-[VH]

The linkers for linking the variable regions of an antibody can bearbitrary peptide linkers that can be introduced by genetic engineering,or synthetic linkers (for example, see Protein Engineering, 9(3),299-305, 1996). However, peptide linkers are preferred in the presentinvention. There are no limitations as to the length of the peptidelinkers. The length can be appropriately selected by those skilled inthe art, depending on the purpose, and is typically 1 to 100 aminoacids, preferably 3 to 50 amino acids, more preferably 5 to 30 aminoacids, and even more preferably 12 to 18 amino acids (for example, 15amino acids).

For example, such peptide linkers include: Ser Gly Ser Gly Gly Ser SerGly Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Gly Gly Gly Ser Ser Gly GlyGly Gly Gly Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly GlyGly Gly Ser Ser Gly Gly Gly Gly Gly Gly (Gly Gly Gly Gly Ser)_(n) (SerGly Gly Gly Gly)_(n)where n is an integer of one or larger. The lengths and sequences ofpeptide linkers can be appropriately selected by those skilled in theart, depending on the purpose.

In an embodiment of the present invention, particularly preferablesc(Fv)2 include the sc(Fv)2 below:

-   [VH]-peptide linker (15 amino acids)-[VL]-peptide linker (15 amino    acids)-[VH]-peptide linker (15 amino acids)-[VL]

Synthetic linkers (chemical cross-linking agents) include cross-linkingagents routinely used to cross-link peptides; for example, N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(succinimidyl)suberate (BS³), dithiobis(succinimidyl propionate) (DSP),dithiobis(succinimidyl propionate) (DTSSP), ethylene glycolbis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST),disulfosuccinimidyl tartrate (sulfo-DST),bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), andbis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). Thesecross-linking agents are commercially available.

In general, three linkers are required to link four antibody variableregions together. The linkers to be used may be of the same or differenttypes. In the present invention, a preferable minibody is a diabody,even more preferably, an sc(Fv)2. Such a minibody can be prepared bytreating an antibody with an enzyme, for example, papain or pepsin, togenerate antibody fragments, or by constructing DNAs encoding thoseantibody fragments and introducing them into expression vectors,followed by expression in an appropriate host cell (see, for example,Co, M. S. et al., 1994, J. Immunol. 152, 2968-2976; Better, M. andHorwitz, A. H., 1989, Methods Enzymol. 178, 476-496; Pluckthun, A. andSkerra, A., 1989, Methods Enzymol. 178, 497-515; Lamoyi, E., 1986,Methods Enzymol. 121, 652-663; Rousseaux, J. et al., 1986, MethodsEnzymol. 121, 663-669; Bird, R. E. and Walker, B. W., 1991, TrendsBiotechnol. 9, 132-137).

Antibodies with extremely high agonistic activity can be prepared byconverting whole antibodies into minibodies.

Modified antibodies for use include antibodies linked to variousmolecules, such as polyethylene glycol (PEG). Alternatively, it is alsopossible to link an antibody to a radioisotope, chemotherapeutic agent,or cytotoxic substance such as a bacterial toxin. Such modifiedantibodies can be prepared by chemically modifying an obtained antibody.Methods for modifying antibodies have been previously established in theart.

Further, antibodies for use in the present invention may be bispecificantibodies. A bispecific antibody may comprise two antigen-binding sitesthat each recognizes different epitopes on a certain molecule.Alternatively, one of the antigen-binding sites may recognize a certainmolecule, and the other may recognize a radioactive substance,chemotherapeutic agent, or cytotoxic substance such as a cell-derivedtoxin. When such cytotoxic substances are used, tumor cell growth can besuppressed by directly adding the cytotoxic substance to cells thatexpress a certain molecule, and specifically damaging the tumor cells.The bispecific antibodies can be prepared by linking pairs of H and Lchains from two types of antibodies, or by fusing hybridomas whichproduce different monoclonal antibodies to produce a fused cellproducing a bispecific antibody. Further, bispecific antibodies can beprepared using genetic engineering techniques.

Antibodies in which sugar chains have been modified can also be used inthe present invention. Techniques for modifying antibody sugar chainshave been previously reported (for example, WO 00/61739 and WO02/31140).

An “antibody” of the present invention includes the antibodies describedabove.

The antibodies expressed or produced as described above can be purifiedby conventional protein purification methods. The antibodies can beseparated and purified, for example, by the combined use of methodsappropriately selected from affinity columns such as a protein A column,column chromatography, filtration, ultrafiltration, salting out,dialysis, and so on (Antibodies A Laboratory Manual. Ed Harlow, DavidLane, Cold Spring Harbor Laboratory, 1988).

The antigen-binding activity of an antibody can be assayed byconventional methods (Antibodies A Laboratory Manual. Ed Harlow, DavidLane, Cold Spring Harbor Laboratory, 1988). For example, anenzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA),radioimmunoassay (RIA), or immunofluorescence method can be used.

The present invention also provides methods of screening for ligandshaving agonistic activity to mutant receptors. In these methods, first,a test substance (test compound) is contacted with a mutant receptor.The “contact” of a test substance with a mutant receptor is typicallyachieved by adding the test substance to a culture medium or extract ofcells expressing the mutant receptor. The methods for achieving contactare not limited to this method. When the test substance is a protein orsuch, the “contact” can be achieved by introducing a DNA vectorexpressing the protein into the cells.

In these methods, the next step comprises detecting signals from themutant receptor. Signals can be detected by the methods described above.

Next, ligands with agonistic activity are selected based on comparisonwith cases when a test substance (control) is not contacted. The ligandsselected in this way are expected to become therapeutic agents fortreating or preventing diseases associated with receptor deficiencies ormutations.

In another embodiment, a screening method of the present inventioncomprises the first step of contacting a test substance with a normalreceptor and measuring agonistic activity. Next, the same test substanceis contacted with a mutant receptor, and agonistic activity is measured.Ligands with high agonistic activity to the mutant receptor as comparedto a normal receptor are then selected.

In still another embodiment, a screening method of the present inventioncomprises the first step of contacting a test substance with a normalreceptor and measuring the agonistic activity. Next, the same testsubstance is contacted with a mutant receptor and agonistic activity ismeasured. Ligands with agonistic activity to both the mutant receptorand normal receptor are then selected.

Measuring agonistic activity in the methods described above can beachieved as described above.

Substances (compounds) obtained by the above-described screening methodsof the present invention are also comprised in the present invention.

Since the ligands of the present invention (for example, antibodies)have agonistic activity, they are expected to be effective therapeuticagents for diseases caused by the impaired response of receptors onwhich the ligands act. Such impaired responses are attributed toreceptor deficiencies or mutations. Specifically, the present inventionprovides therapeutic agents comprising the above-described ligands ofthe present invention, which are used to treat diseases caused by themutant receptors. Representative examples of the diseases describedabove are thrombocytopenia, type II diabetes mellitus, and Laronsyndrome.

A preferred example of a disease of the present invention is congenitalamegakaryocytic thrombocytopenia (CAMT).

When ligands of the present invention or substances (compounds) obtainedby the methods for screening of the present invention are used aspharmaceutical compositions, they can be formulated by methods known tothose skilled in the art. As necessary, the ligands or substances can beused orally, for example, as sugar-coated tablets, capsules, elixirs, ormicrocapsules, or parenterally, as injections of sterile solutions orsuspensions comprising water or other pharmaceutically acceptableliquids. For example, the ligands or the substances can be formulated byappropriately combining with pharmaceutically acceptable carriers orsolvents, specifically, sterile water or physiological saline, vegetableoils, emulsifiers, suspending agents, surfactants, stabilizers,flavoring agents, excipients, vehicles, preservatives, binding agents,and such, and mixing at a unit dosage and form required by acceptedpharmaceutical implementations. In such formulations, the amount of theactive ingredient should be within the required range.

Additives in the tablets or capsules can include, for example, binderssuch as gelatin, corn starch, gum tragacanth, and gum Arabic; excipientssuch as crystalline cellulose; swelling agents such as corn starch,gelatin, and alginic acid; lubricants such as magnesium stearate;edulcorants such as sucrose, lactose, or saccharin; and flavoring agentssuch as peppermint, Gaultheria adenothrix oil, and cherry. When the unitdosage form is a capsule, the above-described materials can alsocomprise a liquid carrier such as oil. A sterile composition forinjection can be formulated using a vehicle such as distilled water usedfor injection, according to standard protocols.

Aqueous solutions used for injections include physiological saline andisotonic solutions comprising glucose or other adjunctive agents such asD-sorbitol, D-mannose, D-mannitol, and sodium chloride. They may also becombined with an appropriate solubilizing agent such as alcohol,specifically, ethanol, polyalcohol such as propylene glycol orpolyethylene glycol, or non-ionic detergent such as polysorbate 80™ orHCO-50.

Oil solutions include sesame oils and soybean oils, and can be combinedwith solubilizing agents such as benzyl benzoate or benzyl alcohol. Theymay also be formulated with buffers, for example, phosphate buffers orsodium acetate buffers; analgesics, for example, procaine hydrochloride;stabilizers, for example, benzyl alcohol or phenol; or anti-oxidants.The prepared injections are typically aliquoted into appropriateampules.

The administration may be carried out orally or parenterally, andpreferably parenterally. Specifically, injection, intranasaladministration, intrapulmonary administration, percutaneousadministration, or such can be used. Injections include intravenousinjections, intramuscular injections, intraperitoneal injections, andsubcutaneous injections. The injection solutions can also besystemically or locally administered. The administration methods can beproperly selected according to the patient's age, condition, and such.When the compounds can be encoded by DNA, the DNA can be inserted into avector for gene therapy, and gene therapy can be carried out. The dosagemay be, for example, in the range of 0.0001 to 1,000 mg/kg body weight.Alternatively, the dosage may be, for example, in the range of 0.001 to100,000 mg/person. However, the dosage is not restricted to the valuesdescribed above. The dosage and administration methods depend on apatient's weight, age, and condition, and can be appropriately selectedby those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the structure of a pCOS2-hMPLfull vector.

FIG. 2 is a diagram showing the structure of a pCOS2-hMPLfullG305Cvector.

FIG. 3 is a diagram showing the structure of a pBACsurf1-HMPL-FLAGvector.

FIG. 4 is a diagram showing the agonistic activity of each of thediabodies and hTPO in pCOS2-HA-Ba/F3. The vertical axis indicatesO.D.450/655 nm and the horizontal axis indicates the concentration.

FIG. 5 is a diagram showing the agonistic activity of each of thediabodies and hTPO in hMPL-Ba/F3. The vertical axis indicatesO.D.450/655 nm and the horizontal axis indicates the concentration.

FIG. 6 is a diagram showing the agonistic activity of each of thediabodies and hTPO in hMPL(G305C)-Ba/F3. The vertical axis indicatesO.D.450/655 nm and the horizontal axis indicates the concentration.

FIG. 7 is a diagram showing the structure of a pCOS2-hMPLfullC769Tvector.

FIG. 8 is a diagram showing the structure of a pCOS2-hMPLfullC823Avector.

FIG. 9 is diagram showing the construction of a TA136 sc(Fv)2 gene.

FIG. 10 is a diagram showing the structure of a pCXND3-TA136 sc(Fv)2vector.

FIG. 11 is a diagram showing the agonistic activity of TA136 db andTA136 sc(Fv)2 in hMPL-Ba/F3 cells. The vertical axis indicatesO.D.450/655 nm and the horizontal axis indicates the concentration.

FIG. 12 is a diagram showing the agonistic activity of TA136 db andTA136 sc(Fv)2 in hMPL(G305C)-Ba/F3 cells. The vertical axis indicatesO.D.450/655 nm and the horizontal axis indicates the concentration.

FIG. 13 is a diagram showing the agonistic activity of TA136 db andTA136 sc(Fv)2 in hMPL(C769T)-Ba/F3 cells. The vertical axis indicatesO.D.450/655 nm and the horizontal axis indicates the concentration.

FIG. 14 is a diagram showing the agonistic activity of TA136 db andTA136 sc(Fv)2 in hMPL(C823A)-Ba/F3 cells. The vertical axis indicatesO.D.450/655 nm and the horizontal axis indicates the concentration.

BEST MODE FOR CARRYING OUT THE INVENTION

Herein below, the present invention will be specifically described usingExamples, however, it is not to be construed as being limited thereto.

EXAMPLE 1 Establishment of a Ba/F3 Cell Line

Several reports have described CAMT patients carrying the G305C (R102P)mutation in their thrombopoietin receptor gene. In this context, anexpression vector for the thrombopoietin receptor gene carrying theG305C (RI 02P) mutation was constructed and introduced into Ba/F3 cellsby the method described below. The prepared DNA fragments were: thenormal thrombopoietin receptor gene (SEQ ID NO: 1) and the mutant genein which the C at nucleotide position 305 from the initiation codon hasbeen substituted for G (SEQ ID NO: 3). These DNA fragments were digestedwith the restriction enzymes EcoRI and SalI, and introduced into theEcoRI-SalI site of the animal cell expression vector pCOS2-Ha to preparepCOS2-hMPLfull (FIG. 1) and pCOS2-hMPLfullG305C (FIG. 2).

After the plasmids pCOS2-hMPLfull, pCOS2-hMPLfullG305C, and as anegative control pCOS2-Ha were treated with PvuI, 20 μg of each plasmidwas transfected into Ba/F3 cells under the conditions described below.The gene was introduced at a cell density of 1×10⁷ cells/ml in PBS usingGENE PULSER II (BIO-RAD) (Gene Pulser Cuvette, 0.4 cm; 0.33 kV; 950 μF).The medium was then changed with RPMI1640 comprising 10% FBS, 1 ng/mlrmIL3 (Pepro tech), 500 μg/ml Geneticin(GIBCO), 100 unit/ml penicillin,and 100 μg/ml streptomycin to select cells. As a result, hMPL-Ba/F3,hMPL(G305C)-Ba/F3, and pCOS2-HA-Ba/F3 cell lines were obtained from therespective vectors described above.

EXAMPLE 2 Preparation of the Extracellular Domain Protein ofThrombopoietin Receptor

To prepare the antigen for producing anti-thrombopoietin receptorantibodies, a system for producing and secreting the extracellulardomain of human thrombopoietin receptor using the insect cell line Sf9was constructed as described below. A gene construct comprising FLAG tagplaced downstream of the extracellular domain of human thrombopoietinreceptor (Gln26-Trp491) was prepared and inserted into the PstI-SmaIsite of pBACsurf-1 (Novagen), to construct pBACsurf1-hMPL-FLAG (FIG. 3).The resulting gene construct (SEQ ID NO: 5) can secrete theextracellular domain of thrombopoietin receptor using a secretory signalsequence derived from baculovirus gp64 protein. 4 μg of the vector wastransfected into Sf9 cells using Bac-N-Blue Transfection Kit(Invitrogen), according to the protocol attached to the kit. After threedays of culture, the culture supernatants were collected and recombinantviruses were isolated using plaque assays. Stock viral solutions wereprepared, and then infected to Sf9 cells. The resulting culturesupernatants were collected, and adsorbed to a Q Sepharose Fast Flowcolumn (Pharmacia). The column was eluted with PBS comprising 500 mMNaCl and 0.01% Tween20. The eluate was adsorbed to M2 Affinity Resin(Sigma). The resins were eluted with 100 mM Glycine-HCl (pH 3.5)comprising 0.01% Tween20. Immediately after elution, the eluate wasneutralized with 1M Tris-Cl (pH 8.0). The resulting solution was treatedby gel filtration chromatography using Superdex 200 26/60 (PBScomprising 0.01% Tween20) to purify the protein.

EXAMPLE 3 Preparation of Anti-Thrombopoietin Receptor Diabody

MRL/lpr mice were immunized seven times with the purified protein of theTPOR extracellular domain. The first immunization was carried out using100 μg of the protein, and subsequent immunizations were each performedusing 50 μg of protein. The immunized cells were fused withP3-X63-Ag8-U1 (P3U1) cells by methods commonly used to preparehybridomas. The hybridomas that produced anti-thrombopoietin receptorantibodies were selected by ELISA assay using the purified protein(VB08B, VB45B, VB033, VB140, and VB157).

Meanwhile, Balb/c mice were immunized a total of 11 times withhMPL-Ba/F3 cells at one-week to five-month intervals. 1.0×10⁷ cells wereintraperitoneally administered to the mice each time. Hybridomas werethen prepared by the same method as described above. The hybridomas thatproduced anti-thrombopoietin receptor antibody were selected (TA136).

The cDNAs for the variable regions of the antibody H and L chains werecloned from each of the hybridomas thus prepared. The cloned cDNAs weresequenced. Based on the nucleotide sequences, genes encoding diabodieswere designed with FLAG tag at their C termini (VB08B db, VB45B db,VB033 db, VB140 db, VB157 db, and TA136 db), and inserted into theexpression vector pCXND3 for animal cells (pCXND3-VB08B db, pCXND3-VB45Bdb, pCXND3-VB033 db, pCXND3-VB140 db, pCXND3-VB157 db, pCXND3-TA136 db).Each prepared vector was introduced into COS7 cells, and the culturesupernatant was collected after three days of culture. The concentrationof diabody in each culture supernatant was determined by BIAcore(Pharmacia) using M2 antibody (Sigma).

EXAMPLE 4 Assay for Diabody Dependency of Ba/F3 Cell Line

pCOS2-HA-Ba/F3 cells, hMPL_Ba/F3 cells, and hMPL(G305C)-Ba/F3 cells wereeach diluted to 2.0×10⁵ cells/ml with medium (RPMI1640 comprising 10%FBS, 100 unit/ml penicillin, and 100 μg/ml streptomycin). The cells werealiquoted (60 μl/well) into the wells of 96-well plates. hTPO (R&D) wasdiluted to a final concentration of 25 μg/ml with CHO-S-SFM II, and thenaliquoted into the wells (40 μl/well). Each diabody/COS7 sup (VB08B db,VB45B db, VB033 db, VB140 db, VB 157 db, and TA136 db) was diluted 1, 3,9, 27, 81, and 243 times using CHO-S-SFM II, and then aliquoted into thewells (40 μl/well). The plates were incubated for 24 hours, and thenCell Count Reagent (nacalai tesque) was added to each well (10 μl/well).The O.D.450/655 nm of each well was measured after two hours of culture.The result showed that the responsiveness of hMPL(G305C)-Ba/F3 cells tohTPO and other agonistic antibodies was markedly decreased. However. TA136 db (SEQ ID NO: 7) was found to exhibit strong agonistic activity tohMPL(G305C)-Ba/F3 cells, while it exhibited weak agonistic activity tohMPL_Ba/F3 cells expressing the normal receptor (FIGS. 4 to 6).

In SEQ ID NO: 8, the amino acid sequence from positions 49 to 54corresponds to heavy chain CDR1; from 69 to 84 corresponds to heavychain CDR2; from 117 to 123 corresponds to heavy chain CDR3; from 163 to174 corresponds to light chain CDR1; from 190 to 196 corresponds tolight chain CDR2; and from 229 to 237 corresponds to light chain CDR3.

EXAMPLE 5 Establishment of a Ba/F3 Cell Line (2)

As in Example 1, expression vectors were constructed for each of themutant thrombopoietin receptor genes carrying the C769T (R257C) mutationand the C823A (P275T) mutation, which were found in some CAMT patients.The constructs were introduced into Ba/F3 cells. Nucleotide T wassubstituted for the nucleotide C at position 769 from the initiationcodon in the thrombopoietin receptor gene (SEQ ID NO: 1), to produce thegene of SEQ ID NO: 9; and nucleotide A was substituted for thenucleotide C at position 823, to produce the gene of SEQ ID NO: 11.These DNA fragments were digested with the restriction enzymes EcoRI andSalI, and introduced into the EcoRI-SalI site of the animal cellexpression vector pCOS2-Ha to obtain pCOS2-hMPLfullC769T (FIG. 7) andpCOS2-hMPLfullC823A (FIG. 8), respectively.

After treating the plasmids pCOS2-hMPLfullC769T and pCOS2-hMPLfullC823Awith PvuI, 20 μg of each of them was transfected into Ba/F3 cells underthe conditions described below. The gene transfer was carried out byelectroporation at a cell density of 1×10⁷ cells/ml in PBS using GENEPULSER II (BIO-RAD) (Gene Pulser Cuvette 0.4 cm; 0.33 kV; 950 μF). Themedium was then changed to RPMI1640 comprising 10% FBS, 1 ng/ml rmIL3(Pepro tech), 500 μg/ml Geneticin (GIBCO), 100 units/ml penicillin, and100 μg/ml streptomycin to select cells. As a result, hMPL(C769T)-Ba/F3and hMPL(C823A)-Ba/F3 cell lines were respectively obtained from thevectors described above.

EXAMPLE 6 Preparation of Anti-Thrombopoietin Receptor Antibody sc(FV)2

TA136 sc(Fv)2 gene was constructed using pCXND3-TA136 db described aboveby the procedure described below (FIG. 9).

PCR was carried out using a combination of primer A(TAGAATTCCACCATGAGAGTGCTGATTCCTTTGTGGCTGTTCACAGCCTTTCCTGGTACCCTGTCTGATGTGCAGCTGCAGG/SEQ ID NO: 15) and primer B(TGGGTGAGAACAATTTGCGATCCGCCACCACCCGAACCACCACCACCCGAACCACCACCACCTGAGGAGACGGTGACTGAGG/SEQ ID NO: 16); and also a combination ofprimer C (CAGTCACCGTCTCCTCAGGTGGTGGTGGTTCGGGTGGTGGTGGTTCGGGTGGTGGCGGATCGCAAATTGTTCTCACCCAGTC/SEQ ID NO: 17) and primer D(ATTGCGGCCGCTTATCACTTATCGTCGTCATCCTTGTAGTCTTTGATTTCCAGCTTGGT G/SEQ IDNO: 18). The resulting PCR products were combined and used as a templatein another PCR using primers A and D. The resulting DNA fragment ofabout 800 bp was digested using the restriction enzymes EcoRI and NotI,and cloned into pBacPAK9 (CLONTECH) to prepare pBacPAK9-scTA136.

PCR was then carried out using pBacPAK9-scTA136 as a template withprimer E (GATGTGCAGCTGCAGGAGTCGGGAC/SEQ ID NO: 19) and primer F(CCTGCAGCTGCACATCCGATCCACCGCCTCCCGAACCACCACCACCCGATCCACCACCTCCTTTGATTTCCAGCTTGGTGC/SEQ ID NO: 20). The resulting DNA fragment ofapproximately 800 bp was cloned into the pGEM-T Easy vector (Promega).

After confirming the nucleotide sequence, the DNA was digested with therestriction enzyme PvuII. The resulting DNA fragment of approximately800 bp was inserted into the PvuII site of pBacPAK9-scTA136 to preparepBacPAK9-TA136 sc(Fv)2. The prepared vector was digested with therestriction enzymes EcoRI and NotI. The resulting DNA fragment ofapproximately 1600 bp was cloned into the expression vector pCXND3 toprepare pCXND3-TA136 sc(Fv)2 (SEQ ID NO: 13; FIG. 10).

EXAMPLE 7 Evaluation of TPO-Like Agonistic Activities of TA1 36 db andTA1 36 sc(Fv)2

The DNA constructs pCXND3-TA136 db and pCXND3-TA136 sc(Fv)2 wereintroduced into COS7 cells. Their respective culture supernatants werecollected after three days of culture. The diabody concentrations in theprepared culture supernatants were determined by BIAcore (Pharmacia)using M2 antibody (Sigma).

hMPL-Ba/F3 cells, hMPL(G305C)-Ba/F3 cells, hMPL(C769T)-Ba/F3 cells, andhMPL(C823A)-Ba/F3 cells were each diluted to 4.0×10⁵cells/ml usingmedium (RPMI1640 comprising 10% FBS, 100 unit/ml penicillin, and 100μg/ml streptomycin). The cells were aliquoted into the wells of 96 wellplates (60 μl/well). 40 μl of hTPO (R&D) and the culture supernatant ofthe COS7 cells described above were added to each well, and the platewas incubated for 24 hours. 10 μl of Cell Count Reagent (Nacalai Tesque)was added to each well. O.D.450/655 nm was determined after incubatingthe plate for two hours.

The results showed that, in all three mutant thrombopoietin receptorcell lines, TA136 sc(Fv)2 exhibited much stronger agonistic activitythan hTPO and TA136 db (FIGS. 12 to 14). Furthermore, TA136 db was foundto show agonistic activity comparable to that of the natural ligand hTPOwhen converted into sc(Fv)2, although in hMPL-Ba/F3 cells expressingnormal thrombopoietin receptor, TA136 db exhibited weaker activity thanthat of hTPO (FIG. 11).

INDUSTRIAL APPLICABILITY

The present invention provides ligands (antibodies) to treat patientswith diseases caused by mutant receptors, for example, CAMT;polynucleotides encoding these antibodies; vectors comprising thepolynucleotides; host cells comprising the vectors; and methods forproducing the antibodies. In addition, the present invention alsoprovides methods for gene therapy using polynucleotides that encode theantibodies. The methods of the present invention provide methods fortreating various genetic diseases caused by mutations in genes thatencode cell membrane proteins. Henceforth, individualized geneticdiagnosis is likely to become widely available for patients. Theantibody engineering techniques of the present invention enable thedevelopment of pharmaceutical agents matched to individual genotypes.

1. A ligand having agonistic activity to a mutant receptor.
 2. Theligand of claim 1, where the ligand is an antibody.
 3. The ligand ofclaim 1, where the ligand has greater agonistic activity to the mutantreceptor than the natural ligand.
 4. The ligand of any one of claim 1,where the mutant receptor is a receptor resulting from a mutation(s) inthe amino acid sequence.
 5. The ligand of any one of claim 1, where themutant receptor has lost responsiveness to the natural ligand.
 6. Theligand of any one of claim 1, where the mutant receptor causes adisease.
 7. The ligand of any one of claim 1, where the mutant receptoris a mutant thrombopoietin receptor.
 8. The ligand of claim 2, where theantibody is a minibody.
 9. The ligand of claim 8, where the minibody isa diabody.
 10. A method for transducing a signal to a mutant receptor bybinding a ligand.
 11. The method of claim 10, where the ligand is anantibody.
 12. The method of claim 10, where the mutant receptor resultsfrom an amino acid mutation(s).
 13. The method of claim 10, where themutant receptor has lost responsiveness to the natural ligand.
 14. Themethod of claim 10, where the mutant receptor is associated with diseaseonset.
 15. The method of claim 10, where the mutant receptor is a mutantthrombopoietin receptor.
 16. A method for treating a disease caused by amutant receptor, by binding a ligand to the mutant receptor.
 17. Themethod of claim 16, where the ligand is an antibody.
 18. A method ofscreening for a ligand having agonistic activity to a mutant receptor,where the method comprises the steps of: (a) contacting a test substancewith the mutant receptor; (b) detecting a signal in the mutant receptor;and (c) selecting a ligand having agonistic activity.
 19. A method ofscreening for a ligand having agonistic activity to a mutant receptor,where the method comprises the steps of: (a) determining agonisticactivity to a normal receptor; (b) determining agonistic activity to themutant receptor; and (c) selecting a ligand having greater agonisticactivity to the mutant receptor than the normal receptor.
 20. A methodof screening for a ligand having agonistic activity to a normal and amutant receptor, where the method comprises the steps of: (a)determining agonistic activity to the normal receptor; (b) determiningagonistic activity to the mutant receptor; and (c) selecting a ligandhaving agonistic activity to both the normal and the mutant receptors.21. The method of claim 18, where the ligand is an antibody.
 22. Asubstance obtained by the method of claim
 18. 23. A therapeutic agentfor a disease caused by a mutant receptor, where the agent comprises aligand of the mutant receptor.
 24. The therapeutic agent of claim 23,where the ligand has agonistic activity to the mutant receptor.
 25. Thetherapeutic agent of claim 23, where the ligand is an antibody.
 26. Thetherapeutic agent of claim 23, where the mutant receptor results from anamino acid mutation(s).
 27. The therapeutic agent of claim 23, where themutant receptor has lost responsiveness to the natural ligand.
 28. Thetherapeutic agent of claim 23, where the mutant receptor is a mutantthrombopoietin receptor.
 29. The therapeutic agent of claim 23, wherethe disease is congenital amegakaryocytic thrombocytopenia.
 30. Theligand of claim 8, where the minibody is sc(Fv)2.
 31. The method ofclaim 19, where the ligand is an antibody.
 32. The method of claim 20,where the ligand is an antibody.